PFTK1 interacts with cyclin Y to activate non-canonical Wnt signaling in hepatocellular carcinoma

doi:10.1016/j.bbrc.2014.05.002

Biochemical and Biophysical Research Communications

Volume 449, Issue 1, 20 June 2014, Pages 163-168

https://doi.org/10.1016/j.bbrc.2014.05.002 Get rights and content

Highlights

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PFTK1 forms a direct complex with cyclin Y.
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PFTK1–cyclin Y complex activates the noncanonical Wnt signaling.
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The Rho GTPases are activated by PFTK1–cyclin Y complex.

Abstract

PFTK1 is a Cdc2-related protein kinase that is frequently upregulated in human hepatocellular carcinoma (HCC) where it correlates with metastatic features and motile phenotypes. To understand the modulated pathway underlining the PFTK1 action, here we show a physical interaction between PFTK1 and cyclin Y (CCNY) in promoting noncanonical Wnt signaling. In HCC cells, we found PFTK1 forms a direct complex with CCNY, and together readily upregulate key components of Wnt signaling (Dvl2 and Naked1). Exogenous expression of PFTK1 and CCNY activated Rho GTPases, which are known targets of the noncanonical path. In line with Rho GTPases activation, we also found marked actin polymerizations in cells with PFTK1–CCNY co-expressions. Our findings highlight a PFTK1–CCNY complex in activating noncanonical Wnt signaling in HCC cells.

Introduction

Wnt protein is a family of secreted glycoproteins that could trigger intracellular signalings through binding with the receptor Frizzled family and other Wnt ligands on cell membrane. The Wnt pathway functions in multiple cellular biological processes and is involved in various human diseases including cancer [1], [2], [3], [4]. The complex Wnt signalings are divided into two branches: the canonical β-catenin-dependent pathway and the noncanonical planar cell polarity (PCP) and Wnt/Ca²⁺ pathways. Canonical Wnt signals activate target genes through stabilization and nuclear translocation of β-catenin, controlling cell proliferation and cell fate determination; while noncanonical Wnt signals involve activation of Rho family of small GTPases and an increase of intracellular Ca²⁺, thereby regulating cell polarity and motility [5].

PFTK1, also named PFTAIRE1, is a member of Cdc2-related serine/threonine protein kinases which share a highly conserved motif PSTAIRE and are crucial regulators of cyclins and cell cycle [6]. Human PFTK1 is highly expressed in the brain, pancreas, kidney, heart, testis and ovary, but low in placenta, lung and the liver [6]. The function of PFTK1 is recently reported as a cyclin-dependent kinase (CDK) regulating cell cycle progression and cell proliferation by specifically interacting with members of cyclin proteins such as cyclin D3 (CCND3) and cyclin Y (CCNY) [7], [8]. Interestingly, the interaction of PFTK1 with CCNY, a cyclin protein with key functions in Drosophila embryogenesis [9], is reported to corporately mediate phosphorylation of low-density-lipoprotein receptor-related protein 6 (LRP6) in Drosophila [8], [9], [10]. It is well known that phosphorylation of transmembrane receptor LRP6 represents an important event and an initial step of the canonical Wnt signaling cascade [11], [12], [13], suggesting a potential role of PFTK1–CCNY complex in controlling the Wnt pathway. However whether this PFTK1–CCNY protein interaction also exists in cancer and its effect on the Wnt pathway in tumor cells remain to be explored.

Hepatocellular Carcinoma (HCC) is the fifth most common cancer and ranks third among causes of cancer-related deaths in men worldwide [14]. It is highly fatal since survival patients from diagnosis is often less than six months and only 5–9% of patients survive over five years [14]. High incidence of recurrence and metastasis is the principal causes of poor prognosis after hepatic resection in individuals with HCC [15]. The elucidation of molecular basis and biological mechanisms by which HCC cells disseminate hence hold much importance in understanding HCC metastasis. Previously our group reported regional chr. 7q21-q22 gain is closely associated with advanced metastatic HCC [16]. PFTK1 was further defined as a target proto-oncogene from this aberrant region [17]. We found common overexpression of PFTK1 in HCC tumor tissue compared to adjacent non-tumoral liver, and more interestingly upregulation of PFTK1 was more profound in cases with microvascular invasion and associated with poorly differentiated tumors [17], [18]. Functionally, PFTK1 could confer cell invasive potentials and cell motilities through actin cytoskeletal polymerization as evident by both ectopic expression and knockdown investigations in HCC cells [17].

Given that HCC is one of the most common cancer type that shows activation of Wnt pathway [19], we thus hypothesis that PFTK1 might control the Wnt signaling pathway in HCC cells via interacting with cyclin Y.

Section snippets

Cell culture

HCC cell lines, HKCI-3 and HKCI-C3, were established from patients who underwent curative surgery for HCC at Prince of Wales Hospital, Hong Kong [20]. These cell lines were cultured in AIM-V medium supplemented with 10% fetal bovine serum, 1× l-glutamine and 1× NEAA (Life Technologies, Carlsbad, CA).

Transfection

Full length cDNA of PFTK1 and CCNY were cloned to pEF6/V5-His and pCMV-Flag-3A vectors, respectively. The authenticity of both constructs was verified by DNA sequencing. PFTK1-pEF6/V5-His and

Protein interaction between PFTK1 and CCNY in HCC cells

We first determined the physical association of PFTK1 and cyclin Y (CCNY) in two HCC cell lines. Full length PFTK1 and cyclin Y were cloned into V5-pEF6 and Flag-pCMV vectors respectively, and cotransfected into HKCI-3 and HKCI-C3 cells both of which express low levels of PFTK1. Forced expression of PFTK1 and CCNY in total lysates was first verified by Western blot using anti-V5 and anti-Flag antibodies, respectively (Fig. 1). Coimmunoprecipitation was then applied to investigate the protein

Discussion

In this study, we demonstrated the presence of protein interaction between PFTK1 and cyclin Y in HCC cells. The PFTK1–CCNY complex activates the β-catenin-independent noncanonical Wnt pathway but keeps the canonical Wnt/β-catenin intact. The subsequent activation of Rho GTPases (RhoA, Rac1 and Cdc42) in the noncanonical Wnt pathways further leads to actin polymerization and stress fibers formation.

Previously Jiang and co-workers identified cyclin Y as a PFTK1 interacting protein by yeast

Acknowledgment

This work was supported by a General Research Fund from the Hong Kong Research Grants Council (Ref. No.: CUHK 476711).

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Cyclin L1- and L2-Cdk11 complexes may play critical roles in centriole duplication, spindle dynamics and sister chromatid cohesion at centromeres during mitosis, although the specific activation by cyclin L was not always clear in these studies [146–148]. Y-type cyclins, which bind Cdk14 and Cdk16 kinases, have been shown to induce Wnt signaling during mitosis through Cdk14-mediated Lrp5/6 phosphorylation [149], and mediate the activation of non-canonical Wnt signaling in hepatocellular carcinoma cell lines [150]. These are only a few representative examples that suggest the complicate relationship between multiple cyclin subfamilies, in many cases poorly characterized, and cell cycle regulation at different levels.
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More and more researches have reported the potential mechanisms. For example, Leung et al. indicates that CDK14 could promote invasion and migration of HCC cells by physical interaction between CDK14 and cyclin Y (CCNY) in promoting noncanonical Wnt signaling [28]. Leung et al discovered that CDK14 enhances HCC cells migration and invasion by inactivating the actin-binding motile suppressing function of TAGLN2 via phosphorylation [27].
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View full text

PFTK1 interacts with cyclin Y to activate non-canonical Wnt signaling in hepatocellular carcinoma

Highlights

Abstract

Introduction

Section snippets

Cell culture

Transfection

Protein interaction between PFTK1 and CCNY in HCC cells

Discussion

Acknowledgment

Cell

Gene

FEBS Lett.

Dev. Cell.

Mod. Pathol.

Cell

Dev. Biol.

Semin. Cell Dev. Biol.

An updated overview on wnt signaling pathways: a prelude for more

Circ. Res.

The wnt signaling pathway in development and disease

Annu. Rev. Cell Dev. Biol.

Wnt signaling pathways meet rho GTPases

Genes Dev.

WNT signaling pathway and stem cell signaling network

Clin. Cancer Res.

Functional characterization of human PFTK1 as a cyclin-dependent kinase

Proc. Natl. Acad. Sci. U. S. A.

Cyclin Y is a novel conserved cyclin essential for development in drosophila

Genetics

LDL receptor-related proteins 5 and 6 in wnt/beta-catenin signaling: arrows point the way

Development

LDL-receptor-related proteins in wnt signal transduction

Nature

Initiation of wnt signaling: control of wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions

Development